CN108977428A - Bromelain enzyme stabilizers and its preparation method and application - Google Patents
Bromelain enzyme stabilizers and its preparation method and application Download PDFInfo
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- CN108977428A CN108977428A CN201811069112.7A CN201811069112A CN108977428A CN 108977428 A CN108977428 A CN 108977428A CN 201811069112 A CN201811069112 A CN 201811069112A CN 108977428 A CN108977428 A CN 108977428A
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
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- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
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- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/22—Cysteine endopeptidases (3.4.22)
- C12Y304/22004—Bromelain (3.4.22.4)
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Abstract
The invention discloses a kind of bromelain enzyme stabilizers and its preparation method and application, are related to field of biotechnology.Bromelain enzyme stabilizers provided by the invention include following component: 0.015-0.13 parts of reducing agent, 0.002-0.07 parts of metal ion chelation agent, 0.01-0.09 parts of organic carboxylate, 0.05-0.15 parts of preservative and 0.001-0.02 parts of vitamin according to parts by weight.The bromelain enzyme stabilizers have many advantages, such as that the half-life period that protective enzyme active fruit is obvious, reduces enzyme activity loses, extends enzyme, usage amount are few and at low cost.In addition, the preparation method is easy to operate the present invention also provides the preparation method and application of bromelain enzyme stabilizers, it is convenient for industrialized production；Bromelain enzyme stabilizers can be added directly for having wide range of applications in the preservation of enzyme solid powder or liquid enzymes production process.
Description
Technical field
The present invention relates to field of biotechnology, in particular to a kind of bromelain enzyme stabilizers and preparation method thereof
And application.
Background technique
Bromelain (Bromelain), alias bromelain are present in pineapple (Anana comosus) plant
Proteolytic enzyme is to extract from pineapple stem end or skin, through one kind obtained from purification, purification, concentration and freeze-drying
Natural plant enzyme.Bromelain is light yellow amorphous powder, and molecular weight is about 3300, isoelectric point 9.55, pH
Between 6.5-7.5, optimal pH 7.1 is incompletely dissolved in water, is also insoluble in ethyl alcohol, chloroform and acetone and other organic solvent.
Bromelain belongs to glycoprotein, containing 2.1% sugared ingredient, there is the aminohexose of 4 N- acetylations, is by dredging
The complex composite object and a kind of sulfydryl proteolytic enzyme of base protein and non-protein enzyme composition, belong to mixture.Bromelain
Enzyme exists as single peptide chain, and in bromelain enzyme molecule, the essential group to play a crucial role to its catalytic activity is thin
Base, amino, trp residue and histidine residues.Because bromelain contains a unstable free thin base, so pineapple
Protease is easily oxidized, and is formed a kind of quinone-mercaptan compound that can voluntarily degrade, is lost activity so as to cause enzyme.Enzyme molecule
Three-dimensional conformation can change under the action of some external force, such as hydrogen bond, Van der Waals force, covalent disulfide bonds, hydrophobic phase interaction
With with electrostatic interaction etc., other factors such as temperature, pH, metal ion, processing aid or activator be will lead under enzyme activity
Drop.
Bromelain inevitably needs to carry out in certain condition in industrial processes as a kind of enzyme preparation
Process, transport and store for a long time such a intermediate links.And under normal temperature state, as a kind of biocatalyst, pineapple
Protease is highly prone to the influence of environmental factor variation and microbial contamination during processing, production, transport and storage etc.；And
And the variation of such as environmental factor temperature, pressure, pH and ionic strength can cause the deactivation of zymoprotein；Furthermore microorganism
Rapid growth then quickly can cause zymoprotein to be biodegradable and inactivate.Therefore, the stability for improving bromelain, subtracts
The stability of enzyme activity loss in few production process, as far as possible holding enzyme during transport, storage is to improve it in industry
Metaplasia produces and the key point of industrial applications.
The thermal stability of enzyme, the research in terms of the shelf life of extension enzyme are improved for enzyme preparation product at present, is mainly had
Four aspect: (1) in thermophilic organisms enzyme research；(2) research of protein denaturation；(3) chemical modification of protein molecule；
(4) protective effect of the addition protective agent to enzyme.And have about the method for improving bromelain enzyme stabilizers: (1) adding related
Food additives reduce the oxidation of enzyme active center by the protective effect to enzyme active center, improve enzyme activity stability；(2)
Bromelain enzyme stability is improved by being chemically modified to bromelain；(3) by the way that bromelain is fixed
Metaplasia changes its resistance to heat rating etc. at immobilised enzymes to improve bromelain enzyme stability.The most commonly used is pass through to be added
Protectant mode protects enzyme.Research protectant for bromelain currently on the market, mainly according to pineapple
Self-characteristic possessed by protease improves bromelain by addition salt, polysaccharide or polyalcohols etc. in conjunction with production
The stability of enzyme enzyme activity increases industrial production value.It is larger as the general usage amount of protective agent using polysaccharide or polyalcohol etc.,
For bromelain medicinal extract or liquid bromelain enzyme solutions, additive amount is bigger, though for single salt protective agent
Certain protective role so can also be played under lesser dosage, but, placement after enzyme unobvious for the protecting effect of enzyme activity
Loss living is still very fast, especially in the preservation of solid powdery bromelain.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide a kind of bromelain enzyme stabilizers, which, which has, protects
The advantages that protecting obvious enzyme activity effect, the loss of reduction enzyme activity and extending the half-life period of enzyme；And each component is cheap, component is simple
It is single, be conveniently easy to get, usage amount is few and it is easy to use, cost is relatively low and facilitates popularization and application；In addition, bromelain enzyme stabilizers can
Directly addition is for having wide range of applications in the preservation of enzyme solid powder or liquid enzymes production process.
The second object of the present invention is to provide a kind of preparation method of bromelain enzyme stabilizers, by each group of formula ratio
Divide and obtain bromelain enzyme stabilizers after mixing, the preparation method is easy to operate, is convenient for industrialized production.
The third object of the present invention is to provide bromelain enzyme stabilizers in reducing the loss of bromelain enzyme activity
Using.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
In a first aspect, the present invention provides a kind of bromelain enzyme stabilizers, it according to parts by weight include following component: also
Former agent 0.015-0.13 parts, 0.002-0.07 parts of metal ion chelation agent, 0.01-0.09 parts of organic carboxylate, preservative 0.05-
0.15 part and vitamin 0.001-0.02 parts.
Further, on the basis of technical solution provided by the invention, bromelain enzyme stabilizers wrap according to parts by weight
Include following component: 0.045-0.1 parts of reducing agent, 0.005-0.04 parts of metal ion chelation agent, organic carboxylate 0.02-0.08
Part, 0.06-0.13 parts of preservative and 0.001-0.01 parts of vitamin.
Further, on the basis of technical solution provided by the invention, bromelain enzyme stabilizers wrap according to parts by weight
Include following component: 0.075-0.0.09 parts of reducing agent, 0.008-0.01 parts of metal ion chelation agent, organic carboxylate 0.03-
0.05 part, 0.08-0.12 parts of preservative and 0.002-0.005 parts of vitamin.
Further, on the basis of technical solution provided by the invention, reducing agent includes D-araboascorbic acid sodium, half Guang of L-
Propylhomoserin, L-cysteine hydrochloride, sodium pyrosulfite, sodium thiosulfate or β-dredge at least one of base ethyl alcohol, and preferably D- is different
Sodium ascorbate and L-cysteine.
Further, on the basis of technical solution provided by the invention, metal ion chelation agent includes ethylenediamine tetra-acetic acid
At least one of disodium, ethylenediamine tetra-acetic acid or sodium polyphosphate, preferably disodium ethylene diamine tetraacetate.
Further, on the basis of technical solution provided by the invention, organic carboxylate includes sodium acetate, sodium oxalate, wine
At least one of stone acid potassium sodium or sodium citrate, preferably sodium acetate.
Further, on the basis of technical solution provided by the invention, preservative includes potassium sorbate, sodium benzoate or second
At least one of sour calcium, preferably potassium sorbate.
Further, on the basis of technical solution provided by the invention, vitamin includes vitamin B, vitamin C or dimension life
At least one of plain E, preferred vitamin B, further preferred Vitamin B9.
Further, on the basis of technical solution provided by the invention, bromelain enzyme stabilizers wrap according to parts by weight
Include following component: 0.01 part of D-araboascorbic acid sodium, 0.075 part of L-cysteine, 0.01 part of disodium ethylene diamine tetraacetate, acetic acid
0.04 part of sodium, 0.1 part of potassium sorbate and 0.002 part of Vitamin B9.
Second aspect, the present invention provides a kind of preparation methods of bromelain enzyme stabilizers, comprising the following steps:
After mixing by the reducing agent of formula ratio, metal ion chelation agent, organic carboxylate, preservative and vitamin
To bromelain enzyme stabilizers.
The third aspect, the present invention provides bromelain enzyme stabilizers to reduce answering in the loss of bromelain enzyme activity
With；
Preferably, the application by the 1.5-3wt% addition bromelain of solid enzyme powder the following steps are included: stablized
Agent；Or, adding bromelain enzyme stabilizers by the 0.1-0.3wt% of liquid enzymes.
Compared with prior art, the invention has the benefit that
(1) bromelain enzyme stabilizers provided by the invention, wherein reducing agent, can be effective by oxidation resistant effect
The free thin base oxidation for preventing bromelain, prevents enzyme from inactivating；Metal ion chelation agent can be formed with polyvalent metal ion can
Soluble metal complexes reduce influence of the metal ion to enzyme activity；Organic carboxylate can influence structure in solvent and it is hydrophobic mutually
Effect, safeguards the active three-dimensional conformation of enzyme molecule, hence it is evident that extend the half-life period of bromelain；Preservative can effectively reduce dirt
The growth for contaminating microorganism, greatly reduces zymoprotein and is biodegradable and inactivates, and extends enzyme and is kept for the active time；Vitamin competition
Property participate in pineapple enzyme solutions in oxygen molecule consumption, reduce the chance that bromelain enzyme active center is oxidized, indirectly rise
To protection bromelain enzyme active center.
By the rational proportion of above-mentioned each component and constituent content, make bromelain enzyme stabilizers that there is protective enzyme active
The advantages that half-life period that fruit is obvious, reduction enzyme activity is lost and extends enzyme；Each component of bromelain enzyme stabilizers is cheap,
Component is simple, be conveniently easy to get, usage amount is few and easy to use, and making bromelain enzyme stabilizers, cost is relatively low, facilitates popularization and application；
In addition, bromelain enzyme stabilizers can directly add for enzyme solid powder save or liquid enzymes production process in, application range
Extensively.
(2) preparation method of bromelain enzyme stabilizers provided by the invention, after mixing by each component of formula ratio
Bromelain enzyme stabilizers are obtained, the preparation method is easy to operate, is convenient for industrialized production.
(3) bromelain enzyme stabilizers provided by the invention prolong the half-life period of bromelain under conditions of 55 DEG C
One times long or more, protecting effect is obvious.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
According to the first aspect of the invention, a kind of bromelain enzyme stabilizers are provided, according to parts by weight include such as
Lower component: 0.015-0.13 parts of reducing agent, 0.01-0.09 parts of organic carboxylate, is prevented 0.002-0.07 parts of metal ion chelation agent
Rotten agent 0.05-0.15 parts and vitamin 0.001-0.02 parts.
Reducing agent can effectively prevent the free thin base oxidation of bromelain, prevent enzyme from losing by oxidation resistant effect
It is living.The typical but non-limiting content of reducing agent is, for example, 0.015 parts by weight, 0.045 parts by weight, 0.075 parts by weight, 0.09 weight
Measure part, 0.1 parts by weight or 0.13 parts by weight.
Metal ion chelation agent can form soluble metal complex compound with polyvalent metal ion, reduce metal ion to enzyme activity
Influence.The typical but non-limiting content of metal ion chelation agent is, for example, 0.002 parts by weight, 0.005 parts by weight, 0.008
Parts by weight, 0.01 parts by weight, 0.04 parts by weight or 0.07 parts by weight.
Organic carboxylate can influence structure in solvent and hydrophobic interaction, safeguard the active three-dimensional conformation of enzyme molecule, bright
The aobvious half-life period for extending bromelain.The typical but non-limiting content of organic carboxylate is, for example, 0.01 parts by weight, 0.03
Parts by weight, 0.05 parts by weight, 0.07 parts by weight or 0.09 parts by weight.
Preservative can effectively reduce the growth of contaminating microorganisms, greatly reduce zymoprotein and be biodegradable and inactivate, prolong
Long enzyme is kept for the active time.The typical but non-limiting content of preservative is, for example, 0.05 parts by weight, 0.07 parts by weight, 0.09
Parts by weight, 0.12 parts by weight, 0.13 parts by weight or 0.15 parts by weight.
Vitamin competitiveness participates in the consumption of oxygen molecule in pineapple enzyme solutions, reduces bromelain enzyme active center by oxygen
The chance of change indirectly plays the role of protecting bromelain enzyme active center.The typical but non-limiting content example of vitamin
For example 0.001 parts by weight, 0.004 parts by weight, 0.008 parts by weight, 0.01 parts by weight, 0.015 parts by weight, 0.018 parts by weight or
0.02 parts by weight.
By the rational proportion of above-mentioned each component and constituent content, make bromelain enzyme stabilizers that there is protective enzyme active
The advantages that half-life period that fruit is obvious, reduction enzyme activity is lost and extends enzyme；Each component of bromelain enzyme stabilizers is cheap,
Component is simple, be conveniently easy to get, usage amount is few and easy to use, and making bromelain enzyme stabilizers, cost is relatively low, facilitates popularization and application；
In addition, bromelain enzyme stabilizers can directly add for enzyme solid powder save or liquid enzymes production process in, application range
Extensively.
In a preferred embodiment, bromelain enzyme stabilizers include following component according to parts by weight: reduction
0.045-0.1 parts of agent, 0.005-0.04 parts of metal ion chelation agent, 0.02-0.08 parts of organic carboxylate, preservative 0.06-
0.13 part and vitamin 0.001-0.01 parts.
In a preferred embodiment, bromelain enzyme stabilizers include following component according to parts by weight: reduction
0.075-0.0.09 parts of agent, 0.008-0.01 parts of metal ion chelation agent, 0.03-0.05 parts of organic carboxylate, preservative 0.08-
0.12 part and vitamin 0.002-0.005 parts.
Content by further optimizing each component in bromelain enzyme stabilizers matches, and bromelain can be made steady
The advantages that determining obvious agent protective enzyme active fruit, the loss of reduction enzyme activity and extending the half-life period of enzyme is more prominent, is conducive to it in enzyme
Popularization and application in solid powder preservation or liquid enzymes production process.
In a preferred embodiment, reducing agent includes D-araboascorbic acid sodium, L-cysteine, L-cysteine
Hydrochloride, sodium pyrosulfite, sodium thiosulfate or β-dredge at least one of base ethyl alcohol, preferably D-araboascorbic acid sodium and L- half
Cystine.
Bromelain is a kind of thiol protease, and the sulfydryl in molecule is one of necessary group of enzyme show activity,
Sulfydryl, which is easily oxidized to form disulfide bond, is suppressed enzymatic activity.It, can be effective by its antioxidation after reducing agent is added
The free thin base oxidation for preventing bromelain, prevents enzyme from inactivating.
The antioxidant effect of reducing agent, preferably D-araboascorbic acid sodium and L- can be preferably played by preferred reducing agent
Cysteine is used in combination, and can further promote reducing agent to prevent the free sulfhydryl groups oxidation of bromelain, more effectively reduce
Enzyme activity loss.
In a preferred embodiment, metal ion chelation agent includes disodium ethylene diamine tetraacetate, ethylenediamine tetrem
At least one of acid or sodium polyphosphate, preferably disodium ethylene diamine tetraacetate.
Plant layout production in, due to mechanical equipment and raw and auxiliary material etc., inevitable bromelain by
The pollution of metal ion, such as Cu2+、Fe2+、Pb2+There is stronger inhibiting effect Deng to bromelain enzyme activity.And metal ion chela
Mixture can reduce influence of the metal ion to enzyme to a certain extent.
Influence of the metal ion to enzyme activity can be more effectively reduced by metal ion chelating agent, is protected to greatest extent
Hold enzyme activity.It is preferred that disodium ethylene diamine tetraacetate is metal ion chelation agent, the effect of metal ion chelation agent can be preferably played
Fruit, enhancing bromelain reduce the effect of enzyme activity loss.
In a preferred embodiment, organic carboxylate includes sodium acetate, sodium oxalate, sodium potassium tartrate tetrahydrate or citric acid
At least one of sodium, preferably sodium acetate.
Hydrophobic interaction plays an important role in maintaining bromelain enzyme molecule three-dimensional conformation, organic in water and water solubility
The in the mixed solvent of object composition, the size of hydrophobic interaction depend on the structure of solvent, when in the mixed solvent hydrone most
When big degree forms hydrogen bond, maximum hydrophobic interaction can get, influence of the polyol to enzyme molecule stability, just
Influence depending on these substances to molecular structure water.
Structure in solvent and hydrophobic interaction can be reduced to the three-dimensional structure of the activity of enzyme molecule by preferred organic carboxylate
The active three-dimensional conformation of enzyme molecule is safeguarded in the influence of elephant, is played bromelain enzyme stabilizers preferably and is reduced enzyme activity loss
Effect.
In a preferred embodiment, preservative includes at least one in potassium sorbate, sodium benzoate or calcium acetate
Kind, preferably potassium sorbate.
Due to the effect of microbial enzyme, especially oxidative dehydrogenation during bromelain is extracted, purifies and is stored
Enzyme can make bromelain that oxidation occur, reduce enzymatic activity.Preservative, which is added, can effectively reduce contaminating microorganisms
Growth, reduce the effect of microbial enzyme, improve the activity of bromelain.
Bromelain stabilizer can be made to play preferably effect by the preservative in preferred bromelain enzyme stabilizers
Fruit more effectively improves enzymatic activity, extends enzyme and is kept for the active time.
In a preferred embodiment, vitamin includes at least one in vitamin B, vitamin C or vitamin E
Kind, preferred vitamin B, further preferred Vitamin B9.
Preferred vitamin B9, also referred to as folic acid, folic acid can the competitive consumption for participating in oxygen molecule in pineapple enzyme solutions, reduce
The chance that bromelain enzyme active center is oxidized indirectly plays the role of protecting bromelain enzyme active center.By excellent
The vitamin type added in bromelain is selected, bromelain can be made preferably to play the effect for reducing enzyme activity loss,
Keep the activity of bromelain.
In a preferred embodiment, a kind of typical bromelain enzyme stabilizers include such as according to parts by weight
Lower component: 0.01 part of D-araboascorbic acid sodium, 0.075 part of L-cysteine, 0.01 part of disodium ethylene diamine tetraacetate, sodium acetate
0.002 part of 0.04 part, 0.1 part of potassium sorbate and folic acid (Vitamin B9).
By the component and constituent content in preferred bromelain enzyme stabilizers, bromelain enzyme stabilizers can be made preferably
The effect for reducing enzyme activity loss is played, the activity of bromelain is improved.
According to the second aspect of the invention, a kind of preparation method of bromelain enzyme stabilizers is provided, including following
Step:
After mixing by the reducing agent of formula ratio, metal ion chelation agent, organic carboxylate, preservative and vitamin
To bromelain enzyme stabilizers.
The each component of formula ratio is obtained into bromelain enzyme stabilizers after mixing, the preparation method is easy to operate, just
In industrialized production.
According to the third aspect of the present invention, bromelain enzyme stabilizers are provided and are reducing the loss of bromelain enzyme activity
In application；
Preferably, the application by the 1.5-3wt% addition bromelain of solid enzyme powder the following steps are included: stablized
Agent；Or, adding bromelain enzyme stabilizers by the 0.1-0.3wt% of liquid enzymes.
Add bromelain enzyme stabilizers in proportion in bromelain finished powder, under 20 DEG C of normal temperature states, spinach
Trailing plants protease solid powder stores 1 year, and enzyme activity retention rate improves 96.5%.This protection is used in liquid bromelain production process
Agent, compared with not having to protectant bromelain production process, enzyme activity can be improved 122.48%, and protecting effect is obvious.
It is used it should be noted that bromelain enzyme stabilizers provided by the invention add after can preparing in proportion,
Use can also be successively added according to the content of wherein each component；Addition can be mixed, can also individually add, specifically make
It can voluntarily be selected with situation.
Bromelain enzyme stabilizers provided by the invention can be applied in bromelain enzyme protection, reduce bromelain
Enzyme activity loss, extends the half-life period of enzyme.It can be by being added in solid enzyme powder and liquid enzymes production process, using wide
It is general.
In order to further appreciate that the present invention, the method for the present invention and effect are done further in detail combined with specific embodiments below
Explanation.Each raw material of the present invention can pass through commercially available acquisition.
Embodiment 1
A kind of bromelain enzyme stabilizers include following components according to parts by weight: 0.005 part of D-araboascorbic acid sodium,
0.01 part of L-cysteine, 0.002 part of disodium ethylene diamine tetraacetate, 0.01 part of sodium acetate, 0.05 part of potassium sorbate and folic acid
0.001 part.
Embodiment 2
A kind of bromelain enzyme stabilizers include following components: 0.04 part of D-araboascorbic acid sodium, L- according to parts by weight
0.09 part of cysteine, 0.07 part of disodium ethylene diamine tetraacetate, 0.09 part of sodium acetate, 0.15 part of potassium sorbate and folic acid 0.02
Part.
Embodiment 3
A kind of bromelain enzyme stabilizers include following components: 0.06 part of D-araboascorbic acid sodium, L- according to parts by weight
0.04 part of cysteine, 0.005 part of disodium ethylene diamine tetraacetate, 0.02 part of sodium acetate, 0.13 part of potassium sorbate and folic acid 0.01
Part.
Embodiment 4
A kind of bromelain enzyme stabilizers include following components: 0.02 part of D-araboascorbic acid sodium, L- according to parts by weight
0.06 part of cysteine, 0.008 part of disodium ethylene diamine tetraacetate, 0.05 part of sodium acetate, 0.08 part of potassium sorbate and folic acid 0.003
Part.
Embodiment 5
A kind of bromelain enzyme stabilizers include following components: 0.01 part of D-araboascorbic acid sodium, L- according to parts by weight
0.075 part of cysteine, 0.01 part of disodium ethylene diamine tetraacetate, 0.04 part of sodium acetate, 0.1 part of potassium sorbate and folic acid 0.002
Part.
Embodiment 6
The present embodiment and the difference of embodiment 5 are: dredging base ethyl alcohol with β-and substitute D-araboascorbic acid sodium.
Embodiment 7
The present embodiment and the difference of embodiment 5 are: substituting disodium ethylene diamine tetraacetate with sodium polyphosphate.
Embodiment 8
The present embodiment and the difference of embodiment 5 are: substituting sodium acetate with sodium citrate.
Embodiment 9
The present embodiment and the difference of embodiment 5 are: substituting potassium sorbate with sodium benzoate.
Embodiment 10
The present embodiment and the difference of embodiment 5 are: substituting folic acid with vitamin E.
Comparative example 1
A kind of bromelain enzyme stabilizers include following components according to parts by weight: 0.001 part of D-araboascorbic acid sodium,
0.15 part of L-cysteine, 0.001 part of disodium ethylene diamine tetraacetate, 0.15 part of sodium acetate, 0.02 part of potassium sorbate and folic acid
0.05 part.
Embodiment 5 is compared, and constituent content is different.
Comparative example 2
A kind of bromelain enzyme stabilizers include following components: 0.15 part of D-araboascorbic acid sodium, L- according to parts by weight
0.001 part of cysteine, 0.1 part of disodium ethylene diamine tetraacetate, 0.005 part of sodium acetate, 0.2 part of potassium sorbate and folic acid 0.0005
Part.
Embodiment 5 is compared, and constituent content is different.
Comparative example 3
A kind of bromelain enzyme stabilizers include following components according to parts by weight: 0.01 part of disodium ethylene diamine tetraacetate,
0.04 part of sodium acetate, 0.1 part of potassium sorbate and 0.002 part of folic acid.
Compared with Example 5, lack D-araboascorbic acid sodium and L-cysteine.
Comparative example 4
A kind of bromelain enzyme stabilizers include following components: 0.01 part of D-araboascorbic acid sodium, L- according to parts by weight
0.075 part of cysteine, 0.04 part of sodium acetate, 0.1 part of potassium sorbate and 0.002 part of folic acid.
Compared with Example 5, lack disodium ethylene diamine tetraacetate.
Comparative example 5
A kind of bromelain enzyme stabilizers include following components: 0.01 part of D-araboascorbic acid sodium, L- according to parts by weight
0.075 part of cysteine, 0.01 part of disodium ethylene diamine tetraacetate, 0.1 part of potassium sorbate and 0.002 part of folic acid.
Compared with Example 5, lack sodium acetate.
Comparative example 6
A kind of bromelain enzyme stabilizers include following components: 0.01 part of D-araboascorbic acid sodium, L- according to parts by weight
0.075 part of cysteine, 0.01 part of disodium ethylene diamine tetraacetate, 0.04 part of sodium acetate and 0.002 part of folic acid.
Compared with Example 5, lack potassium sorbate.
Comparative example 7
A kind of bromelain enzyme stabilizers include following components: 0.01 part of D-araboascorbic acid sodium, L- according to parts by weight
0.075 part of cysteine, 0.01 part of disodium ethylene diamine tetraacetate, 0.04 part of sodium acetate and 0.1 part of potassium sorbate.
Compared with Example 5, lack folic acid.
Comparative example 8
A kind of Bromelain stabilization protective agent includes the following ingredient of mass percent: lysolecithin 40%, sugarcane
Sugar 10%, dextrin 30% and skimmed milk power 20%.
Comparative example 9
A kind of Bromelain stabilization protective agent includes the following ingredient of mass percent: isomalt
3.3%, Macrogol 600 1.2%, soluble starch 2.5%, calcium chloride 2% and deionized water 91%.
For the effect for further verifying above-described embodiment and comparative example, ad hoc following experimental example.
1 enzyme activity determination of experimental example
To the bromelain stabilizer that embodiment 1-6 and comparative example 1-9 are provided keep the effect of bromelain enzyme activity into
Row detection.
One, experiment reagent
1, enzyme dilution:
Weigh L-cysteine hydrochloride (C3N7NO2S·HCl·H2O=157.63) 5.27g adds water 500mL to dissolve, separately
Take disodium ethylene diamine tetraacetate (C10H14N2O8Na2·2H2O) 2.23g adds water 200mL to dissolve, and two liquid of merging are uniform, uses
0.1mol/L sodium hydroxide solution or 0.1mol/L hydrochloric acid solution are adjusted to pH=5.5, are diluted with water to 1000mL.To the greatest extent may be used using preceding
Energy Fresh, 2~8 DEG C of storages are no more than three days.
2,0.05mol/L disodium phosphate soln:
Weigh disodium hydrogen phosphate (Na2HPO4·12H2O) 17.89g is dissolved in water, and is settled to 1000mL, 2~8 DEG C of storages
It deposits.
3, solution of trichloroacetic acid:
Trichloroacetic acid 17.99g is weighed, anhydrous sodium acetate 29.94g and glacial acetic acid 18.9mL are added, after adding suitable quantity of water to dissolve, is added
Water is settled to 1000mL, and shaking uniformly, can be used 1 month under being sealed.
4, casein substrate solution (face with now match):
It weighs and is dried in silica gel drier to the casein 2.5g of constant weight, set in beaker, 0.05mol/L phosphoric acid hydrogen is added
Two sodium solution 40mL.It heats while stirring in boiling water bath, until being completely dissolved, after cooling, is adjusted to pH with 1.0mol/L hydrochloric acid
=7.0, it is transferred in 50mL volumetric flask, adds water to scale.
5, enzyme solution (500U/mL)
It weighs and is set in beaker through bromelain 0.5g, the dissolution of 40mL distilled water, stirring is added, until be completely dissolved, it is cold
But it after, is transferred in 50mL volumetric flask, adds water to scale.
Two, experimental method
1, enzyme activity defines (U/g)
Refer at 37 DEG C of temperature, pH value 7.0, enzyme amount needed for caseinhydrolysate per minute generates 1 μ g tyrosine.
2, Specification Curve of Increasing
A: sample sets
A. 860 μ L kaliumphosphate buffers (20mM, pH=7.0) are added into 2mL centrifuge tube；
B. the 40 μ L (20U) of bromelain enzyme solution for adding fresh configuration, is placed in 37 DEG C of water-baths and keeps the temperature 10min；
C. the casein substrate that 100 μ L (5mg) are kept the temperature in advance is added, accurately reacts 10min in 37 DEG C of water-baths；
D. 1000 μ L solution of trichloroacetic acid are added into centrifuge tube, concussion shakes up；
E. it is centrifuged 5min at 13000rpm, takes supernatant 400 μ L to 96 orifice plates, in microplate reader (275nm,
Enterpoint light absorption value) is measured, is returned to zero by blank of water.
B: control group
A. 860 μ L kaliumphosphate buffers (200mM, pH=7.0) are added into 2mL centrifuge tube；
B. the 40 μ L (20U) of bromelain enzyme solution for adding fresh configuration, is placed in 37 DEG C of water-baths and keeps the temperature 10min；
C. 1000 μ L solution of trichloroacetic acid are added into centrifuge tube, react 10min in 37 DEG C of water-baths；
D. the casein substrate that 100 μ L (5mg) are kept the temperature in advance is added, concussion shakes up；
E. it is centrifuged 5min at 13000rpm, takes supernatant 400 μ L to 96 orifice plates, in microplate reader (275nm,
Enterpoint light absorption value) is measured, is returned to zero by blank of water.
C: standard curve
Take 0.02mg/L, 0.04mg/L, 0.06mg/L, 0.08mg/L, 0.10mg/L, 0.12mg/L, 0.16mg/L,
0.18mg/L and 0.2mg/L tyrosine standard solution dilution, makees reference with aqueous solution, and solution is measured at wavelength 275nm
Absorbance, standard curve is drawn according to absorbance and corresponding tyrosine concentration.
3, enzyme activity determination
Bromelain finished product enzyme (1,000,000 U/g) is configured to the bromelain enzyme solutions in every mL solution containing 500U,
Embodiment 1-6 and comparative example 1-9 the bromelain stabilizer provided are added by 10wt% again, is placed at 55 DEG C and heats, respectively
The bromelain enzyme activity at 1h, 4h and 8h time point is measured, is control not add any protectant bromelain enzyme solutions,
On the basis of liquid initial enzyme activity, the remaining enzyme activity of relative time is calculated.
Three, experimental result
1, absorbance and corresponding tyrosine concentration calibration curve formula: y=6.9254x-0.0046, R2=0.9994.
2, the bromelain stabilizer that embodiment 1-6 and comparative example 1-9 is provided keeps the effect of bromelain enzyme activity to survey
It is fixed that the results are shown in Table 1:
Table 1
Table 1 the results showed that control group (not adding any enzyme stabilizers) with the extension of heating time, pineapple egg
White enzyme enzyme activity is gradually reduced, and when being heated to 8h, enzyme activity is completely lost.Prove that bromelain is unstable at such a temperature, it is high
Temperature destroys bromelain catalytic active center, and half-life period is in 2.5h or so.From the point of view of test results, when with heating
Between extension, the decline of bromelain enzyme activity is slow, it was demonstrated that bromelain enzyme stabilizers carry out bromelain enzyme active center
Good protection.
Embodiment 5 is a preferred embodiment of the present invention, and the difference of embodiment 1-4 and embodiment 5 is constituent content
Difference, but constituent content is within protection scope of the present invention；Embodiment 6-10 from the difference of embodiment 5 is that component is different, but
Component is also all within protection scope of the present invention；The bromelain enzyme activity determination result of embodiment 5 is than embodiment 1-4 and reality
The result for applying a 6-10 is more preferable, it was demonstrated that embodiment 5 is a more preferred embodiment.
The remaining enzyme activity of three times measurement of embodiment 1-7 is all higher than comparative example 1-7；Comparative example 1-2 and 5 phase of embodiment
It is constituent content difference than, difference, the constituent content of comparative example 1 and 2 is not within protection scope of the present invention；Comparative example 3-7
Compared with Example 5, difference is component difference, than embodiment 5 few one or two kind of component.This shows pineapple egg provided by the invention
The effect that each component of white enzyme and the reasonable pairing bromelain enzyme stabilizers of constituent content reduce enzyme activity loss has important shadow
It rings.
The remaining enzyme activity of three times measurement of embodiment 1-7 is all higher than comparative example 8 and comparative example 9, and comparative example 8 and 9 is
The bromelain enzyme stabilizers provided in existing patent, this shows bromelain enzyme stabilizers protection pineapple egg provided by the invention
The effect of white enzyme enzyme activity is better than the bromelain enzyme stabilizers that comparative example 8 and comparative example 9 provide.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
1. a kind of bromelain enzyme stabilizers, which is characterized in that according to parts by weight include following component: reducing agent 0.015-
0.13 part, 0.002-0.07 parts of metal ion chelation agent, 0.01-0.09 parts of organic carboxylate, 0.05-0.15 parts of preservative and dimension
Raw 0.001-0.02 parts of element.
2. bromelain enzyme stabilizers described in accordance with the claim 1, which is characterized in that according to parts by weight include such as the following group
Point: 0.045-0.1 parts of reducing agent, 0.005-0.04 parts of metal ion chelation agent, 0.02-0.08 parts of organic carboxylate, preservative
0.06-0.13 parts and vitamin 0.001-0.01 parts；
Preferably, the bromelain enzyme stabilizers include following component: reducing agent 0.075-0.0.09 according to parts by weight
Part, 0.008-0.01 parts of metal ion chelation agent, 0.03-0.05 parts of organic carboxylate, 0.08-0.12 parts of preservative and vitamin
0.002-0.005 parts.
3. bromelain enzyme stabilizers according to claim 1 or 2, which is characterized in that the reducing agent includes D- different anti-
Bad hematic acid sodium, L-cysteine, L-cysteine hydrochloride, sodium pyrosulfite, sodium thiosulfate or β-are dredged in base ethyl alcohol extremely
Few one kind, preferably D-araboascorbic acid sodium and L-cysteine.
4. bromelain enzyme stabilizers according to claim 1 or 2, which is characterized in that the metal ion chelation agent packet
Include at least one of disodium ethylene diamine tetraacetate, ethylenediamine tetra-acetic acid or sodium polyphosphate, preferably disodium ethylene diamine tetraacetate.
5. bromelain enzyme stabilizers according to claim 1 or 2, which is characterized in that the organic carboxylate includes second
At least one of sour sodium, sodium oxalate, sodium potassium tartrate tetrahydrate or sodium citrate, preferably sodium acetate.
6. bromelain enzyme stabilizers according to claim 1 or 2, which is characterized in that the preservative includes sorbic acid
At least one of potassium, sodium benzoate or calcium acetate, preferably potassium sorbate.
7. bromelain enzyme stabilizers according to claim 1 or 2, which is characterized in that the vitamin includes vitamin
B, at least one of vitamin C or vitamin E, preferred vitamin B, further preferred Vitamin B9.
8. bromelain enzyme stabilizers according to claim 1 or 2, which is characterized in that according to parts by weight include as follows
Component: 0.01 part of D-araboascorbic acid sodium, 0.075 part of L-cysteine, 0.01 part of disodium ethylene diamine tetraacetate, sodium acetate 0.04
Part, 0.1 part of potassium sorbate and 0.002 part of Vitamin B9.
9. a kind of preparation method according to the described in any item bromelain enzyme stabilizers of claim 1-8, which is characterized in that packet
Include following steps:
The reducing agent of formula ratio, metal ion chelation agent, organic carboxylate, preservative and vitamin are obtained into spinach after mixing
Trailing plants albumen enzyme stabilizers.
10. a kind of described in any item bromelain enzyme stabilizers of claim 1-8 or bromelain as claimed in claim 9
The bromelain enzyme stabilizers that the preparation method of stabilizer is prepared are reducing the application in the loss of bromelain enzyme activity；
Preferably, the application is the following steps are included: add bromelain enzyme stabilizers by the 1.5-3wt% of solid enzyme powder；
Or, adding bromelain enzyme stabilizers by the 0.1-0.3wt% of liquid enzymes.
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| CN109875055A (en) * | 2019-04-12 | 2019-06-14 | 重庆骄王天然产物股份有限公司 | Broken juice device and broken juice system |
| CN111019929A (en) * | 2019-12-24 | 2020-04-17 | 福州三合元生物科技有限公司 | Long-acting biological enzyme heat stabilizer and micro-channel continuous synthesis method thereof |
| CN114699517A (en) * | 2022-04-25 | 2022-07-05 | 南京鲸力医药科技有限公司 | Bromelain preparation and application thereof in detumescence composition |
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| CN109875055A (en) * | 2019-04-12 | 2019-06-14 | 重庆骄王天然产物股份有限公司 | Broken juice device and broken juice system |
| CN111019929A (en) * | 2019-12-24 | 2020-04-17 | 福州三合元生物科技有限公司 | Long-acting biological enzyme heat stabilizer and micro-channel continuous synthesis method thereof |
| CN114699517A (en) * | 2022-04-25 | 2022-07-05 | 南京鲸力医药科技有限公司 | Bromelain preparation and application thereof in detumescence composition |
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